Wood diseases caused by fungi are presently the most serious threat to the lifespan of commercial fruit trees and vines throughout the globe. They endanger the profitability and sustained production by decreasing yield and fruit quality, mainly by inducing the decay and death of infected plants. Key species affected by such diseases include grapevine (Vitis spp. and their hybrids), kiwi-fruit (Actinidia spp. and their hybrids), although it is increasingly recognised that similar diseases affect many other hosts, as will be discussed below.
In grapevine (Vitis spp. and hybrids thereof), esca, Petri disease, Eutypa dieback, and black dead arm (BDA) are the main wood diseases inducing such decay and death of the vine plants (Luque et al., 2009, “Symptoms and fungi associated with declining mature grapevine plants in northeast Spain”, Journal of Plant Pathology 91, 381-390). As no effective control measures are available against these diseases, they are dramatically affecting the durability of the wine-growing heritage (Larignon et al., 2009, “Esca et Black Dead Arm: deux acteurs majeurs des maladies du bois chez la vigne”, Comptes Rendus Biologies 332, 765-783.).
Esca is a complex grapevine wood disease that is achieving an unprecedented and increasing worldwide importance. First believed as affecting only mature grapevine, the disease is now found at high levels in much younger plants. It is accepted that esca comprises a number of distinct diseases and that the main causal agents (mostly vascular fungi) invade the vines not only through wounds in the field but also as a result of nursery practices. When infection starts in the nursery, plants may develop different diseases, ranging from Petri decline (also known as “black goo”) to full-blown esca, with or without white decay of the woody tissues.
Esca is caused by the presence of at least three fungi, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Fomitiporia mediterranea, acting alone, in combination or in succession. The two first fungi are responsible for vascular necroses, which appear as dark wood streaking, sometimes with some areas of dark and hard necroses, while F. mediterranea (and/or other wood-rotting basidiomycetes) induces white rot, or “amadou”.
However, the pattern of symptoms and the initial onset of their expression are highly variable: severely infected plants can remain asymptomatic in successive years for a number of years without visible (leaf) symptoms of esca (“hidden” esca). This intermittent pattern of symptom expression leads infected plants to be indistinguishable from healthy ones.
It is of paramount importance to ensure healthy propagation of grapevine in the nursery, where only material without wood streaking should be selected. Once planted in the vineyard, infected propagating material develops Petri disease or decline, also characterised by wood dark streaking, slow dieback and “black goo”. Because these fungi grow inside the wood of trunk and large wooden branches, one cannot reach them by foliage spraying with fungicides. The best remedy still is digging up the infected vine and burning it.
The greatest problem of esca derives from its initial stages: the plants are usually externally asymptomatic, although occasionally fruiting bodies and spores could be present. However, they are capable of disseminating the disease through common vineyard practice, such as pruning. In other words, a typical vineyard machine may pass on the disease from one infected, but asymptomatic, vine to the next ones. Spores are also disseminated by the wind and rain. Thus, while pruning tools and vineyard specialized machinery are not important in spore dissemination, they may play a role in spreading fungal mycelia from diseased plants to healthy ones. However, in what spore dissemination and penetration is concerned, pruning wounds and other wounds caused by vineyard specialized machinery are particularly important. For all these reasons, it is of paramount importance to develop methodologies capable of nondestructive screening and detection of the initial stages of esca, either in nurseries or in vineyards. Improved strategies and methods for treating such fungal infections at an early stage whilst eradication of the disease is still possible are also required.
Plant species other than grapevine are also subject to wood affecting, esca-like diseases. In 1999, a wood decay of kiwifruit vines (Actinidia) was noted for the first time in Italian vineyards. A similar disease was subsequently reported in Greece and New Zealand. Such diseases were described under the names of “esca-like” in France and “Elephantiasis” in Italy.
Analysis of kiwifruit vineyards exhibiting visual symptoms of esca-like disease revealed the presence of two types of necrosis in the wood: a) hard, brown decay initiating in the medular tissues and expanding gradually in the entire trunk or branches, and b) white and soft decay like “amadou wood” of grapevine. Isolations from necrotic parts of wood revealed a number of fungi, including Fomitiporia mediterranea, Phaeoacremonium aleophilum, Phaeoacremonium parasiticum, and Cadophora malorum. At least two of the above fungal species (F. mediterranea and P. aleophilum) are also involved in esca disease of grapevine, and aspects in common between the decay of kiwifruit and esca of grapevine can be hypothesized.
Citrus species (such as the orange cv. Washington navel, lemon and the common mandarin grafted on sour orange rootstocks) are experiencing a serious decline caused by F. mediterranea, in southern Greece orchards. Affected trees exhibit leaf chlorosis, defoliation, and death of shoots and twigs. Cross-sections of the trunks and large branches reveal a light-coloured rot in the centre, which is surrounded by brown hard necrotic wood. Symptoms start from pruning wounds and extend to the rootstock wood, resembling the esca wood symptoms of grapevine.
As well as grapevine, kiwi and citrus, esca-like diseases have also been identified in a number of other species. Phaeoacremonium aleophilum has been reported in Prunus species showing dieback symptoms in the Western Cape, South Africa. Serious dieback symptoms, caused by Pm. parasiticum, have been observed in cherry trees in Greece. Phaeoacremonium mortoniae was shown to cause brown wood staining of Fraxinus pennsylvanica and Salix spp.
One of the key causative agents, Fomitiporia mediterranea, is also found on Corylus avellinus, Olea europaea, Lagerstroemia indica, Actinidia sinensis, Acer negundo, Olea europaea, Quercus sp., Quercus ilex, Ligustrum vulgare and Robinia pseudoacacia. 
A further plant disease, and similarly affecting woody species is Eutypiose, also known as “Eutypa dieback”, is caused by the fungus Eutypa lata. E. lata is a vascular pathogen and occurs on at least 88 species of woody dicots (Carter, 1991. “The status of Eutypa lata as pathogen”, Phytopathological Paper 32) and is responsible for significant economic damage mainly to grapevine and apricot.
Many forest and ornamental species also host the pathogen, but it is not certain whether all isolates are pathogenic, although pathogenicity of E. lata has been confirmed for grapevine, apricot, sweet cherry (Prunus avium L.), chokecherry (Prunus virginiana L. var. demissa), sour cherry (Prunus cerasus L.), almond (Prunus dulcis), apple (Malus domestica Borkh.), pear (Pyrus communis L.), walnut (Jugfans regia L.), olive (Olea europaea L.), and Ceanothus L. spp.
On grapevine, the most recognized symptom of Eutypa dieback is the stunted appearance of shoots at the early growth season, with small, chlorotic and distorted leaves, and short internodes. However, the symptomatic expression fluctuates markedly from year to year. Non-destructive techniques for diagnosis of Eutypa dieback are still unavailable. Furthermore, no efficient treatment is available, and infected plants will die within a few years.
A further disease of concern, especially in grapevine (Vitis) is associated with infection by Botryosphaeriaceae fungi. These have a wide distribution and are commonly associated with dieback and cankers of woody and non-woody host plants, including forest trees, e.g. Pinus spp., Eucalyptus spp., Quercus spp., Olea, stone fruit trees, Protea and grapevine.
On grapevine, several species in Botryosphaeriaceae are associated with wood necrosis and they are also able to infect both young and mature tissues as well as green shoots causing cankers, vascular discoloration, and/or otherwise dark streaking of the wood.
Some Botryosphaericaeae species are responsible for Black Dead Arm (BDA) disease, in which the wood symptoms include V-shaped necroses, similar to those caused by Eutypa lata, and longitudinal brown streaking along the affected tissues. Other symptoms resembling Eutypa dieback, such as stunted chlorotic shoots, deformed leaves with necrotic areas are frequently observed.
It will be appreciated that all these diseases share common characteristics: (1) they are predominantly diseases of wood plants, and especially woody fruit-bearing plants of commercial importance; (2) they are fungal in origin, and (3) the pathogens invariably colonise the vasculature of the plant, especially the xylem. Furthermore, because (4) the disease symptoms are often localised—at least in the early stages—to the interior of the woody parts of the plant, non-invasive diagnosis of the conditions is difficult, if not impossible. In addition, once detected (5) treatment of the disease is difficult, because the internalisation of the fungal infection renders it barely susceptible to external application of fungicides.
In the wider context of diseases of the woody parts of commercially-important plants, one of the greatest problems facing agriculture is the fight against pathogens and insects that develop within the wood and so in a location where conventional fungicides/insecticides do not reach. It is therefore of great importance to develop methods to detect the existence, and the location, within infected plants, of wood diseases during the initial but asymptomatic and contagious phases of the infection (and thus, during a potentially treatable phase) and to develop treatment regimes that are effective against such diseases.
It is amongst the objects of the present invention to address these problems.